Leishmania tarentolae can be used as an easy-to-handle microfactory for the rapid production of viral antigens to face emerging epidemics. We engineered L. tarentolae to express the SARS-CoV-2 receptor-binding domain (RBD) and we recorded the ability of the purified RBD antigen to detect SARS-CoV-2 infection in human sera, with a sensitivity and reproducibility comparable to that of a reference antigen produced in human cells. This is the first application of an antigen produced in L. tarentolae for the serodiagnosis of a Coronaviridae infection. On the basis of our results, we propose L. tarentolae as an effective system for viral antigen production, even in countries that lack high-technology cell factories.To detect and prevent emerging epidemics, discovery platforms are urgently needed, for the rapid development of diagnostic assays. Molecular diagnostic tests for COVID-19 were developed shortly after the isolation of SARS-CoV-2. However, serological tests based on antiviral antibody detection, revealing previous exposure to the virus, required longer testing phases, due to the need to obtain correctly folded and glycosylated antigens. The delay between the identification of a new virus and the development of reliable serodiagnostic tools limits our readiness to tackle future epidemics. We suggest that the protozoan  Escherichia coli; Saccharomyces cerevisiae; E. coli, are expected to produce non-glycosylated proteins, which would then potentially result in altered patterns in antibody detection, when used in the serological diagnosis of viral diseases. The limitations in the diagnostic performances of viral antigens produced in microbial prokaryotes are unfortunate because expression systems such as E. coli are easily engineered, cultured in optimized conditions in bioreactors, and very effective for high yield protein production at low cost  for constitutive, secreted expression. Cloning results in the incorporation of a C-terminal 6xHis-tag onto the resulting Spike fragment, which comprises the RBD and SD1 domains . The pLEXSY-sat2 vector integrates into the chromosomal 18S ribosomal RNA (ssu) locus of the L. tarentolae parasite. In addition, a signal sequence from Leishmania mexicana, which allows the secretion of the target protein into the culture medium, was added. The plasmid was cloned and propagated in E. coli, and then, the plasmid was linearized through digestion with SwaI enzyme. The host L. tarentolae–P10 was then transfected with the linearized plasmid by electroporation, according to the procedures of the manufacturer. Engineered strains were cultured in Brain Heart Infusion (BHI) liquid medium supplemented with porcine hemin , penicillin–streptomycin , and Nourseothricin  (100 μg/ml) at 26°C in the dark under aerated conditions. For strain maintenance, cultures were diluted into fresh BHI medium twice a week.The sequence of SARS-CoV-2 Spike receptor-binding domain (RBD-SD1) was derived from the genomic sequence of the isolated virus “Severe acute respiratory syndrome coronavirus 2 Wuhan-Hi-1” released in January 2020, number MN908947 and comprises 819 nucleotides  and 273 amino acids. The gene coding for RBD-SD1 was codon-optimized for Leishmania cultures were centrifuged 10 min at 3,000 g. Clarified supernatants were filtered using a 0.22-μm nitrocellulose membrane and concentrated at 5,000 g for 30 min, using an Amicon ultracentrifugal filter with a molecular weight (MW) cutoff of 10 kDa. Samples were diluted in a loading buffer 4X (Thermo Fisher Scientific), boiled for 5 min, and subsequently loaded onto a 4–20% gradient polyacrylamide gel (Bio-Rad Laboratories). After electrophoresis, proteins were transferred onto a nitrocellulose membrane (Bio-Rad Laboratories), according to the standard protocols, before blocking for 5 min at room temperature with EveryBlot Blocking Buffer (Bio-Rad Laboratories) and incubation with a 1:3,000 dilution of anti–6xHis-tag–horseradish peroxidase (HRP) antibody (Thermo Fisher Scientific) in the EveryBlot Blocking Buffer for 1 h. After three washes with phosphate-buffered saline (PBS) + 0.1% (v/v) Tween 20 (PBS-T), the membrane was incubated for 5 min with the Clarity Western ECL Substrate (Bio-Rad Laboratories) and detected using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories).Expression of the target protein was evaluated by analyzing a sample of the supernatant from 10 recombinant clones of Lt-RBD by Western blotting. After 72 h of growth in BHI complete medium, supplemented with NTC at 26°C, Leishmania cultures (2.5 L) were centrifuged for 10 min at 3,000 g, and the supernatant was filtered on a 47-mm-diameter activated carbon filter (Merck) (1 filter/0.5 L) to help remove the hemin from the growth media, before a second filtration step on a 0.22-μm nitrocellulose membrane (Merck). The supernatant was concentrated to approximately 100 ml using the Vivaflow 200 system (Sartorius) and compatible PES Vivaflow 200 cassette , at 4°C, before dialysis, using a dialysis membrane with a MW cutoff of 14–16 K (SpectaPor). Dialysis was carried out at 4°C in 2.6-L Binding Buffer . The dialysate was transferred into fresh buffer (lacking Tween 20) after 4 h and then, after a third exchange, overnight at 4°C. Lt-RBD was purified on a 5-ml HisTrap Excel column (Cytiva), pre-equilibrated with Binding Buffer. The supernatant was loaded at a flow rate of 1 ml/min using AKTA basic fast protein liquid chromatography (FPLC) (Cytiva) and washed with Binding Buffer unit until the A280 nm reached baseline. Elution was carried out using a slow, stepwise  imidazole gradient, generated by mixing Binding Buffer with Elution Buffer , eluting at each step with four column volumes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, fractions containing the purified protein were pooled and concentrated to 500 μl, using an Amicon Ultra Filter unit with a MW cutoff of 10,000 (Millipore), and further purified by size exclusion chromatography on a Superdex 200 Increase 10/300 GL (Cytiva) column, pre-equilibrated in 1 × PBS (Merck) at a flow rate of 0.5 ml/min at room temperature. Peak fractions (1 ml per fraction) were pooled and aliquoted for conservation at −80°C. Protein concentration was measured spectrophotometrically using a NanoDrop, using the theoretically calculated A280 nm for 1 mg/ml Lt-RBD of 1.077 and with the BCA Protein Assay Kit (Merck).Recombinant strains expressing Lt-RBD were cultured for 4 days in complete BHI supplemented with NTC (100 μg/ml), Pen-Strep, and hemin (1.25 μg/ml) at 26°C in the dark and in constant agitation under aerated conditions. The The expression of purified Lt-RBD was evaluated both by Coomassie staining and Western blotting, as described above. In particular, the proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) that were incubated for 5 min at room temperature with EveryBlot Blocking Buffer (Bio-Rad Laboratories) and then incubated with a 1:3,000 dilution of anti-SARS/SARS-CoV-2 Coronavirus Spike Protein (subunit 1) antibody (Thermo Fisher Scientific) in the blocking buffer for 1 h. After three washes with PBS-T, the membrane was incubated with a HRP-conjugated immunoglobulin G (IgG) anti-rabbit 1:30,000 (Thermo Fisher Scientific) in the blocking buffer for 1 h. Finally, after three washes with PBS-T, the membrane was incubated for 5 min with the Clarity Western ECL Substrate (Bio-Rad Laboratories) and detected by ChemiDoc Touch Imaging System (Bio-Rad Laboratories).–1 with concomitant buffer exchange with fresh PBS to remove imidazole using Amicon centrifugal filters (Merck), flash-frozen in liquid nitrogen, and kept at -80°C until usage. Before analysis, the protein samples were thawed and subjected to gel filtration using a Superdex 200 10/300 increase column (Cytiva) equilibrated with 25 mM HEPES/NaOH, 150 mM NaCl, pH 7.2 , we recombinantly produced RBD in human cells (hu-RBD), to simulate the viral protein produced during a natural infection in humans. The pCAGGS plasmid for production of the C-terminal His-tagged SARS-CoV-2 Spike RBD (#NR_52310) was obtained from BEI Resources . Recombinant SARS-CoV-2 Spike RBD was produced using HEK293-F cells (Invitrogen) cultivated in suspension using FreeStyle medium (Invitrogen) as described in , pH 7.2 .–1 in PBS using a high-pressure liquid chromatography system (Shimadzu Prominence). For MW characterization, light scattering was measured with a miniDAWN MALS detector (Wyatt), connected to a differential refractive index detector (Shimadzu RID-20A) for quantitation of the total mass, and to a UV detector (Shimadzu SPD-20A), for evaluation of the sole protein content. Chromatograms were collected and analyzed using the glycoconjugate analysis algorithm available in the ASTRA7 software . Calibration of the instrument was verified by injection of 10 μl of monomeric bovine serum albumin (3 mg/L) (Sigma-Aldrich).Recombinant SARS-CoV-2 RBDs hu-RBD and Lt-RBD (20 μl of 1 mg/ml) were injected into a Protein KW-802.5 analytical size-exclusion column (Shodex) and separated with a flow rate of 1 ml minDSF assays on recombinant SARS-CoV-2 Spike RBD samples (hu-RBD and Lt-RBD) at a concentration of 1 mg/ml in PBS buffer were performed using a Tycho NT.6 instrument (NanoTemper Technologies GmbH). Data were analyzed and plotted using the GraphPad Prism 7 (Graphpad Software).For the setup and standardization of the in-house ELISA test (see below), we relied on the human sera reported in For the titration of IgG-specific antibodies, we relied on 10 commercially available human sera, both from symptomatic and asymptomatic donors, positive for the SARS-CoV-2 antibody response (CUST-BB-19032021-1A and CUST-BB-19032021-1B-NeoBiotech) and five pre-pandemic sera (year 2015) kindly provided by the University of Siena as negative controls. We further performed a comparative analysis to assess the validity of the in-house Lt-RBD ELISA on a cohort of 80 human sera collected from asymptomatic subjects in March/April 2020, provided by the University of Milan ] .Before starting the ELISA assay qualification experiments, a series of preliminary assays were performed to select the optimal antigen concentration. To this aim, coating concentrations of 1, 2, 3, and 4 μg/ml were tested against human positive and negative sera for SARS-CoV-2 and compared with Spike-RBD  expressed and purified from HEK293 cells.450) using a SpectraMax ELISA plate  reader.The setup and qualification experiments, as well as antibody titration and the comparative analysis, were performed as described below. ELISA plates were coated with purified recombinant Lt-RBD (2 μg/ml). After overnight incubation at 4°C, coated plates were washed three times with ELISA washing solution (300 μl per well) containing tris-buffered saline (TBS)–0.05% (v/v) Tween 20 (TBS-T) and then blocked for 1 h at 37°C with a solution of TBS containing 5% (w/v) non-fat dry milk . Human serum samples were heat-inactivated at 56°C for 1 h to reduce the risk of the presence of live virus in the sample. Subsequently, twofold serial dilutions, starting from 1:100 in TBS-T containing 5% NFDM, were performed up to 1:51,200. Plates were washed three times, as previously; then, 100 μl of each serial dilution was added to the coated plates and incubated for 1 h at 37°C. Next, after the washing step, Goat anti-human IgG-Fc HRP-conjugated antibody  were added. Plates were incubated at 37°C for 30 min. After incubation, plates were washed, and 3,3’,5,5’-tetramethylbenzidine (100 μl per well) substrate  was added and incubated in the dark at room temperature for 20 min. The reaction was stopped by adding 100 μl of ELISA stop solution  and read within 20 min at optical density at 450 nm  using the above ELISA protocol and was expressed as arbitrary units. Samples for the comparative analysis on the 80 human sera were assayed with a 1:100 dilution and the ODAll samples were tested with a starting dilution of 1:100 in four repetitions per plate and in two different plates, to generate four reportable (RP) values per sample. The percentage of geometric standard deviation (%GSD) was calculated between the RP values (RP) [GSD% = (eSD-1) × 100], where SD is the standard deviation between the natural logarithm (ln) results of the RP values.Precision of the assay was assessed by testing positive control as neat and pre-diluted 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128 in dilution buffer; thus, considering the sample in first well is always diluted (1:100), the effective samples starting dilution in the first well will be 1:100 1:200, 1:400, 1:800, 1:1,600, 1:3,200, 1:6,400, and 1:12,800. The above mentioned sample dilutions will be tested eight times in two different plates from one operator on day 1 (four repetitions per plate); the RP value will be calculated between two determination results from the two different plates. The same testing activity will be performed by another operator on day 2 to obtain a total of eight RP values for each dilution .Linearity was evaluated by performing a linear regression analysis of the base-2 logarithm serum dilution against the base-2 logarithm of the geometric mean titer (GMT) of all the eight RP values of the precision experiments using the method of least squares. The coefficient of determination, y-intercept, and slope of the regression line were calculated and reported.The accuracy of the test was evaluated using the RP values obtained for the evaluation of the precision. According to International Conference on Harmonisation (ICH) guideline Q2 , the accPlates are incubated for 30 min (standard condition) with detection antibody to detect IgG specifically; to assess the influence of this incubation time, plates were incubated with the dilution antibody for 30 min at 37°C and two other different times: 20 and 40 min.The positive and negative controls were tested with a starting dilution of 1:100 in four repetitions, in two plates in two different days for each condition to obtain four RP values.L. tarentolae culture medium, as confirmed by SDS-PAGE and Western blotting using an anti–His-tag antibody . The protein migrated by SDS-PAGE as a single band of approximately 35 kDa, corresponding to its estimated MW; this band was visible for most of the tested clones  expression and purification, via affinity chromatography and size exclusion chromatography . Large-scale expression of Lt-RBD resulted in a yield of approximately 2.3 mg/L. The protein was identified by Western blotting using a specific anti-RBD antibody  were cloned into the pLEXSY-sat2 vector, as described in the “Materials and Methods” section. The selected sequence encodes for the RBD domain (273 amino acids) of the SARS-CoV-2 Spike protein and also includes the SD1 subdomain of subunit S1. The inclusion of the SD1 portion was based on the evidence that this fragment is highly immunogenic . Lt-RBD d clones . The supantibody  and judgantibody .SEC-MALS analysis showed that the purified protein is monodisperse with a total MW of 36.5 kDa, which is 5 kDa greater than the MW predicted on the basis of amino acid composition, due to the glycan moieties . MoleculThe difference between the molecular masses of the two proteins is mainly due to the inclusion of the SD1 subdomain in Lt-RBD. Furthermore, their unfolding temperatures, determined by DSF analysis, are highly similar , indicating that Lt-RBD is as stable as the hu-RBD protein .The RBD-ELISA qualification assay was carried out, as described by The specificity was determined as the ability of the assay to differentiate between similar analytes and particularly to differentiate the target analyte from non-target analytes . To deteThe negative sample, as the heterologous sample, showed negative titers in all performed measurements of this testing series. The RP values for the sample mixtures Pos-Neg and Pos-Het samples showed a GSD% of 15.09%, which meets the acceptance criterion set at ≤ 50%. The positive homologous serum sample showed positive results across eight measurements in total. The negative human serum sample showed negative values, with the starting ODs (1:100 dilution) under the cutoff value established for this validation. Overall, the results reported in The precision of an analytical procedure is generally defined as the standard deviation or relative standard deviation (coefficient of variation) of a series of measurements . The staRepeatability  shows the precision of the assay when the test is carried out in a laboratory over a relatively short time period using the same operator and equipment. Repeatability is assessed by evaluating variation of replicates .The %GSD between four RP values from each operator (in one experiment on the same day) from the precision experiment was calculated and reported for each dilution, for each operator. The negative sample provided negative titer results. The positive sample showed GSD% of < 50% for all dilutions . These rThe purpose of intermediate precision is to determine the capacity of the assay to provide reproducible results when random events occur. Variations could include operators, equipment, different days, and reagents. The intermediate precision was evaluated by performing two different assays by different operators using different sessions of analysis in the same laboratory. The %GSD between the GMT of the four RP values from each operator from the precision experiment was calculated and reported for each dilution. The positive sample showed GSD% of < 50% for all dilutions, which indicates that the assay meets the criteria for intermediate precision .R2 was close to 1 (0.995), whereas the absolute value of the slope was 1.090 , corresponding to the closest dilutional point of 100 and 26,500, respectively . AlthougL. tarentolae, fulfilled all the acceptability criteria. In particular, the assay demonstrated to be specific, reproducible, precise, linear, accurate, and robust. Thus, the procedure is defined as reliable and valid for the serological detection of SARS-CoV-2 IgG-specific antibodies.The robustness gives an indication of the assay reliability when events could occur during testing in a single laboratory. Plates are incubated for 20, 30, and 40 min to assess the influence of this incubation time with HRP. For the three different conditions of incubation time for antibody detection, the %GSD of the results for the positive control was 6.72%, which meets the acceptance criterion set at < 50%, and the negative control showed negative results; hence, the results indicate that the assay is robust . The quar = 0.98 (95% CI 0.95–0.99)]. Moreover, the slope of a major axis regression  was not significantly different from 1 [estimate = 0.98 (95% CI 0.84–1.15)], and the intercept was not significantly different from 0 [estimate = 0.07 (95% CI -0.49–0.63)], indicating a strictly direct proportionality between the antibody titers obtained with the two methods  was evaluated testing 15 human sera, among which ten positive for SARS-CoV-2. As negative controls, five pre-pandemic (2015) sera were included in the assay. The IgG antibody titers were calculated, and the results obtained with the two proteins were compared. As shown in gression  of com-R methods .450 was very close to the cutoff ]. However, the OD450 of Lt-RBD  was significantly lower than those of the com-RBD  , and the slope of a major axis regression of com-RBD on Lt-RBD OD450  was significantly larger than 1 [estimate = 1.13 (95% CI 1.06–1.20)]; the elevation was also significantly larger than 0 [estimate = 0.19 (95% CI 0.19–0.25)]. These statistics indicate that Lt-RBD provided slightly but consistently lower OD450 values compared to the com-RBD, possibly because of the slightly higher MW of the Lt-RBD-SD1 protein, which may affect the performance of the ELISA assay  according to the pre-defined cutoff values . The onle cutoff . From thSA assay .L. tarentolae expression system have been assayed for the serological diagnosis of Leishmania infections in humans and dogs, with successful results both in terms of sensibility and sensitivity , antigens produced in L. tarentolae showed satisfactory performances in serological assays, for both African and American trypanosomiases  the model antigen that we selected for this study, i.e., the RBD protein fragment from SARS-CoV-2, was effectively produced and purified and demonstrated to possess folding and glycosylation patterns comparable to that of the same antigen produced in mammalian cells; (ii) all of the tests carried out to validate an in-house ELISA assay based on Lt-RBD met the established recommendation criteria; (iii) titrations on standard COVID-19–positive sera showed linearity in the recorded values, and a perfect match was observed in the attribution of the sera to the two reference classes (positive or negative), using Lt-RBD or com-RBD; (iv) finally, the analysis with Lt-RBD on 80 sera from a previous study led to an almost perfect match with the results obtained with com-RBD (with just one mismatch).The success in the serodiagnostic use of antigens produced in L. tarentolae, the risk for a viral contamination of the cultures cannot be excluded, although there is currently no evidence for viral infections in this system, and the transmission of viruses from humans and animals to Leishmania cells can be regarded as highly improbable (Leishmania system produces moderate quantities of proteins (up to 30 mg/L; L. tarentolae and humans are very similar (e.g., L. tarentolae was investigated in recombinant human EPO and was demonstrated to be highly homogeneous, with a biantennary glycosylation profile that shares its core with that typical of mammals (L. tarentolae (L. tarentolae proteins is limited (Leishmania and human cells cannot be excluded. However, our results indicate that the RBD antigen produced in L. tarentolae guarantees a reliable serodiagnosis of SARS-CoV-2 infection, in agreement with previous results obtained on HEV (The gold standard for serological diagnosis viral infections in humans and other mammals is the use of antigens produced in mammalian cells, ideally in cells form the same species . Howeverprobable . Certainar e.g., , minor dar e.g., ; in addiar e.g., . N-glyco mammals . Howeverrentolae . In addi limited . In summd on HEV .L. tarentolae as a system that is easy to manipulate and culture, providing a sound alternative for the rapid production of serodiagnostic proteins, even at the forefront of novel epidemics, e.g., in the presence of “spillover” events in tropical countries (Leishmania cells can be obtained in flasks or simple bioreactors, within 2–4 days from the starting of the culture (L. tarentolae holds the potential to be scaled to the industrial level, growing the parasites in bioreactors and harvesting proteins using high throughput strategies (Finally, our study underlines ountries . For exa culture , whereas culture . We emphrategies .L. tarentolae is expected to ensure the production of viral antigens whose glycosylation pattern mimics that determined after expression in mammalian cells (L. tarentolae are the production of proteins with mammalian-like PTMs and the solubility of the proteins (L. tarentolae presents possible disadvantages compared to prokaryotic expression systems: the engineering and the selection of transformed clones is more laborious; yield and production costs are not competitive with those ensured by well-established systems such as E. coli (In addition, in comparison with yeast and insect cells, an cells  while inproteins . Of cour E. coli .The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the University of Milan, approval number 17/20, approval date March 6, 2020. The patients/participants provided their written informed consent to participate in this study.Leishmania maintenance. IV-B, FD, and VB performed the set-up and serological assay. AM contributed to the design of the serological assays and analyzed the data. DR performed the statistical analyses. LG, PG, FF, and SF performed the biochemical analyses. IV-B, FD, and AM wrote the first draft of the manuscript. GZ, EM, SE, and CB revised the manuscript. All authors have read and agreed to the published version of the manuscript.CB and SE conceived and coordinated the study. IV-B and BB contributed to cell culture and The antigen Lt-RBD and its potential application have been described in the patent application no. IT 02021000004160; publication date 23 February 2021. AM, FD, and EM were employed by VisMederi Srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A hydrophobic derivative of ciprofloxacin, hexanoylated ciprofloxacin (CPF-hex), has been used as a photoinitiator (PI) for two-photon polymerization (2PP) for the first time. We present, here, the synthesis of CPF-hex and its application for 2PP of methacrylate-terminated star-shaped poly , as well a systematic study on the optical, physicochemical and mechanical properties of the photocurable resin and prepared three-dimensional scaffolds. CPF-hex exhibited good solubility in the photocurable resin, high absorption at the two-photon wavelength and a low fluorescence quantum yield = 0.079. Structuring tests showed a relatively broad processing window and revealed the efficiency of CPF-hex as a 2PP PI. The prepared three-dimensional scaffolds showed good thermal stability; thermal decomposition was observed only at 314 °C. In addition, they demonstrated an increase in Young’s modulus after the UV post-curing  bone). Moreover, using CPF-hex as a 2PP PI did not compromise the scaffolds’ low cytotoxicity, thus they are suitable for potential application in bone tissue regeneration. The two-photon polymerization 2PP) technology allows real 3D printing at a sub-100-nm resolution PP techno. Due to There are two crucial components in a photocurable resin for 2PP: a photoinitiator (PI) and a monomer (or an oligomer). At the early stages of the 2PP method development, one-photon PIs, such as Irgacure 369, Irgacure 184, etc., were usually used due to their commercial accessibility . These cCompounds with a low fluorescence quantum yield are preferred for 2PP, as this leads to higher population of the active triplet state for initiating the polymerization. It has been shown, recently, that the presence of a carbonyl group in PI results in the reduction of the fluorescence quantum yield due to its strong electron-withdrawing effect, as well as facilitation of the intersystem crossing, owing to the efficient spin-orbital coupling ,16. FollFurthermore, one of this study’s objectives was to assess the effect of UV post-processing on the mechanical properties of three-dimensional (3D) scaffolds based on the investigated compositions. The photocurable resins for 2PP are usually viscous, and polymerization occurs within a small volume of a material called a voxel. Therefore, in a scaffold there are unreacted double bonds trapped between voxels, and the distribution of the components is inhomogeneous . That isBased on the above, this study is divided into three parts . First, 2 column for chromatographic purification. Appropriate fractions were combined and dried using a rotary evaporator that resulted in 90 mg (69% yield) of a slightly yellowish solid.Ciprofloxacin hydrochloride donated by Zhejiang LangHua pharmaceutical Co., Ltd.   was mixed with 1 mL of dimethyl sulfoxide , 0.5 mL of pyridine  and hexanoic anhydride  at room temperature followed by heating up to 50–60 °C during 5 min. Then, the mixture was allowed to cool down to room temperature, and 10 mL of benzene  was added dropwise, resulting in the crude product precipitation. The precipitate was dissolved in acetone  using sonication  and then placed to a SiO1H NMR  δ (ppm): 8.7 , 7.97–7.95 , 7.35–7.34 , 3.84–3.70 , 3.54 , 3.34–3.25 , 2.36 , 1.64 , 1.38–1.30 , 1.2 , 0.88   δ (ppm): −    Rf: 0.95 vs. 0.10 for ciprofloxacin.CPF-hex (1-cyclopropyl-6-fluoro-7-(4-hexanoylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid),   . 19F NMR ; ESI-MS osition) ; TLC  (Mn(GPC) = 3528 g/mol, Mw/Mn = 1.29) synthesized according to the methods described in our previous study . The phoThe scaffolds were produced using a microfabrication femtosecond laser system , which delivers 200-fs pulses at a 70 MHz repetition rate. In order to assess the optimal processing window of the resins, spatially defined structures b, 3D scaUV-Vis absorption spectra were recorded with a Cary-50 spectrophotometer  at a concentration of 800 μg/mL.Fluorescence spectra were recorded with a Fluoromax Plus fluorescence spectrometer . The relative fluorescence quantum yields were measured using anthracene (quantum yield = 0.27 in ethanol) as a reference standard .−1 and averaged over 8 scans. The initial star-shaped methacrylate functionalized polylactide and the scaffolds after 2PP were analyzed using a GladiATR single-reflection accessory  equipped with a monolithic diamond crystal . Each spectrum was calibrated by the intensity of the C–O stretching vibration (1084 cm−1) and underwent the Fourier-transform smoothing procedure by four points.IR spectra were acquired with a FT-IR Spectrum Two spectrometer  in the ATR mode with a resolution of 4 cmTo estimate the optimal power of the 2PP method and the completeness of washing from the uncured photosensitive composition, a KOZO XJF900  microscope and SEM with a Phenom Pro X.  instrument were used.Differential scanning calorimetry (DSC) measurements were performed using an STA 6000 simultaneous thermal analyzer . Samples for DSC experiments (about 10 mg) were encapsulated in standard PerkinElmer pans and heated in a nitrogen medium at a gas flow rate of 20 mL/min and a linear heating rate of 10 °C/min.2. The local mechanical characteristics for the initial scaffolds and for the irradiated scaffolds were measured with a Piuma Nanoindenter . A cantilever with the spring constant of 126.1 N/m and the tip radius of 56 µm was used. The measurements were performed in distilled water at 37 °C, the scan size was 1 × 1 mm2 with a 100 µm step by the X and Y axes. Using the Hertzian model, we calculated the effective Young’s modulus and created distribution maps of the Young’s moduli over the surface.To study the mechanical characteristics, the square scaffolds of side width 3 mm and height 400 μm were formed. The scaffolds were additionally irradiated for 10 min with a UV light-emitting diode  with a wavelength of 365 nm at the intensity of 3.9 mW/cmThe material cytotoxicity was assessed using Live/Dead  staining and AlamarBlue  and PicoGreen  assays. We used mesenchymal stromal cells (MSC) derived from the gingiva. The cells were cultured as described elsewhere  and moni1H, 19F NMR and mass spectrometry (ESI-MS) (see The N-hexanoylated ciprofloxacin derivative (CPF-hex) was synthesized by the reaction of ciprofloxacin with 1.5 equivalents of hexanoic anhydride . The str-MS) see .The introduction of a hydrophobic group into ciprofloxacin increased its lipophilicity. The calculated lipophilicity values of ciprofloxacin and its hexanoylated derivative were 1.78 and 3.26, respectively. As compared with unmodified ciprofloxacin, CPF-hex showed greater solubility in dichloromethane and THF – suitable solvents for polylactide a,b. At tn-π* states, e.g., with acceptor carbonyl groups in their structure, were synthesized. Similar to many photoinitiators of radical polymerization, ciprofloxacin has a carbonyl group, which is directly bound and conjugated to the aromatic system. According to the simplified mechanism of photoactivation of such compounds, it may generate a biradical upon absorption of light, due to the homolytic cleavage of one of the π–π bonds. Further intramolecular processes and intermolecular interactions lead to the destruction of the piperazine cycle, defluorination and decarboxylation. The transition of fluoroquinolones to the excited state is generally described as a transition from the S0 state to the excited S1 state, then, to the triplet T1 state by intersystem crossing. The radiative decay from the S1 state to S0 is responsible for the fluorescence (with the emission maximum at 450 nm), while the excitation energy transfer to oxygen molecules is provided by the T1 state [Note that the linear absorption coefficient of CPF-hex at the 2PP wavelength was higher than that of Bis-b. Many studies on the synthesis of 2PP PIs focus on the development of compounds with high nonlinear absorption coefficients or large two-photon absorption cross-sections. To measure them, the Z-scan technique is used. However, the efficiency of 2PP is complex and multifactorial, with several processes taking place over different time scales. For example, a high fluorescence yield can lead low 2PP efficiency, since relaxation of the excited state via fluorescence and transition to the triplet state are competing pathways . To reduT1 state . The genIn practical terms, all the factors are summed up in the processing or fabrication window, which is related to the intensity of the laser radiation. The processing window is defined by two thresholds ,33. The Several approaches are used to evaluate the processing window and the photoinitiation efficiency of 2PP PIs, such as single-line writing or the fabrication of individual test structures. In this study, the efficiency of CPF-hex as a 2PP PI was evaluated by the fabrication of a three-dimensional structure consisting of seven cylinders a. The thA photocurable resin, based on methacrylate-terminated star-shaped polylactide and Bis-b as a photoinitiator, was used as a reference standard due to good results we had obtained previously . In thisThe fluorescence spectra for scaffolds with CPF-hex and Bis-b were recorded  and theiCiprofloxacin, being a representative quinolone carboxylic acid derivative, generally shows a complex fluorescence behavior depending on the solvent polarity and pH. It has native fluorescence emission at 440 nm (quantum yield = 0.28) upon excitation at 278 nm in water .The excitation and emission maxima of scaffolds with CPF-hex were 325 and 380 nm, respectively b. The reScaffolds with Bis-b c had the2v symmetrical anthraquinone derivatives with the fluorescence quantum yield up to 0.043. In [2v-symmetrical 2PP PIs were studied (the emission quantum yields = 0.2). The structuring tests revealed the efficiency of these materials as 2PP PIs.Low fluorescence quantum yields are desirable for 2PP PIs, since fluorescence is a common loss channel, competing with the initiation of polymerization. The fluorescence quantum yields of CPF-hex and Bis-b  in chloroform were determined to be 0.079 and 0.003, respectively. Similar values of the fluorescence quantum yield of 2PP PIs have been obtained in other studies. For example, the authors of  synthesi.043. In , other sIt can be concluded that, although fluorescence-based techniques are prone to larger errors, when the fluorescence quantum yield is very small , the results show that the ciprofloxacin derivative exhibits a low fluorescence quantum yield. This finding, as well as the intense self-absorption and relatively broad processing window, might make CPF-hex a promising candidate as a photoinitiator for 2PP.−1), bending vibrations of CH3 (1452 and 1380 cm−1), stretching vibrations of C–O–C (bands in the range from 1046 to 1183 cm−1), etc. [−1, assigned to the stretching vibrations of the terminal methacrylate groups (blue section in −1 (stretching vibration of carbonyl groups in unsaturated esters) to the loss of C = C groups  for the scaffold is less intense than that for the initial methacrylate-terminated star-shaped polylactide and the position of the rocking vibrations of CH2 increases from 741 to 748 cm−1, which may be attributed to the changes in the polymer network (red sections in −1) , etc. . At the g) equal to 59.7 and 56.1 °C, respectively  in the photocured system are observed, therefore, Tg decreases.In our experiment, the change in the polarity of the polymer system most probably is a decisive factor in terms of the influence on Tg decrease after the cross-linking, the presence of the unreacted composition in the sample, which plays a role of a distinctive plasticizer, may also be mentioned.Note also that, in the first scan , exotherThe obtained thermogravimetry results are presented in Appropriate mechanical properties are one of the key requirements of three-dimensional scaffolds for their subsequent in vitro and in vivo applications. Scaffolds need to maintain the structural integrity after implantation in the host tissue until regeneration. The scaffold’s mechanical properties will affect the biomaterial’s degradability, cell behavior and signaling pathways ,43.In the case of 2PP-fabricated scaffolds, their mechanical properties are significantly determined by the composition of the photocurable resin and the laser fabrication parameters. Both factors affect the degree of double bond conversion and, hence, the elastic modulus of the scaffold. In our work, we studied the effect of UV irradiation on the local elastic modulus of scaffolds. It was shown that the more pronounced increase in mechanical properties after the UV post-curing was observed for scaffolds based on methacrylate-terminated star-shaped polylactide and CPF-hex: the Young’s modulus increased by 40%, from 336 ± 79 MPa to 564 ± 183 MPa. For scaffolds with Bis-b, in contrast, the UV post-treatment did not lead to a significant growth of the elastic modulus, while the spread of values increased . Note, tWe have previously shown  that thrAfter the seeding, we showed that MSC attached to a scaffolds’ surface and actively proliferated . The celIn this study, we have developed a simple approach for the preparation of an efficient and non-toxic photoinitiator for 2PP, which consists in the introduction of a hydrophobic group into ciprofloxacin, a known commercial antibiotic. This simple modification allows achieving excellent solubility of this compound (CPF-hex) in the photocurable composition. In addition, the photophysical studies showed that CPF-hex exhibited high absorption at the two-photon wavelength, a low fluorescence quantum yield (0.079) and a relatively broad processing window. These properties make CPF-hex a promising candidate for application as a photoinitiator in 2PP printing. Finally, well-defined three-dimensional scaffolds were fabricated, characterized by good thermal stability, no cytotoxicity and a high Young’s modulus.


Escherichia coli is the single most important causative agent for this condition. Colistin has recently been classified as a critically important antimicrobial for human health, as it is a last-resort drug against certain multi-drug-resistant Gram-negative bacteria. Therefore, the use of colistin has been significantly reduced in some countries, including Denmark. Despite this, the drug is still commonly used to treat diarrhea in pigs in many countries, and there is a need to understand the risks associated with this practice. We performed a prospective cohort study to investigate the effect of colistin treatment on the changes in the average minimum inhibitory concentration (MIC) in commensal E. coli in a pig herd where no colistin-resistant bacteria were detectable before treatment. One group of pigs was batch treated with colistin after the clinical observation of diarrhea, one group was batch treated with colistin approximately 10 days before the expected onset of diarrhea, and a control group was not treated with colistin but provided with nonantimicrobial antidiarrheal feed supplement. Treatment with colistin in the dose and time combinations used did not result in a significant increase in the average colistin MIC values in E. coli. Moreover, no E. coli strains showed a MIC above the breakpoint of >2 mg/L against colistin. Co-selection of resistance to other antimicrobials was not observed.The treatment of diarrhea in the postweaning period is a common reason for the use of antimicrobials in pig production, and  Antimicrobial resistance (AMR) is considered a threat to human health. Worldwide, at least 700,000 people die annually from infection caused by AMR bacteria, and if appropriate actions are not taken, it has been estimated that by the year 2050, this number may reach up to 10 million, and the cost will be up to USD 100 trillion annually , and theE. coli in weaner pigs, and for several decades, it has been a frequent choice of drug for this indication in pigs . Th. ThE. conc oxide 00 ppm foData on the growth of individual pigs were recorded 3–7 days after the pigs entered the nursery unit and again on Day 42. In addition, individual and batch treatments for diarrhea, dead pigs, and other health data were recorded at departure from the piglet barn.From each pen, a composite fecal sample was collected on a sterile sock, as described in a previous study , by walk−6. Two technical replicates were prepared for each sample. Twenty microliters of each dilution were plated onto different agar plates by the drop plate method [Coliform bacteria was carried out on MacConkey agar (MCA) , and the quantification of presumptive drug-resistant Coliform bacteria was carried out on MacConkey agar supplemented with 2 µg/mL colistin sulfate (MCA + Col), 16 µg/mL tetracycline (MCA + Tet), 16 µg/mL ampicillin (MCA + Amp) and 2 µg/mL cefotaxime (MCA + Cef) antimicrobials as previously reported [E. coli, recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [E. coli colonies. For statistical purposes, the counted colony numbers were converted into log10 CFU per gram of feces.The fecal materials from each of the sock samples were scraped off carefully. One gram of fecal sample was diluted in 9 mL of phosphate-buffered saline (PBS) to make a 1:10 dilution, which was then tenfold serially diluted up to 10e method . Quantitreported ,28. The (EUCAST) . The agaE. coli colonies (n = 105) were collected and stored at −80 °C from the colistin-added agar plates for further analysis. The species identity of randomly picked colonies (n = 600) from MCA containing the different antibiotics was confirmed to be E. coli by matrix-assisted laser desorption ionization-time of flight mass spectrometry .All presumptive colistin-resistant E. coli (n = 105) colonies isolated from colistin-containing MacConkey agar plates were subjected to the broth microdilution (BMD) method to determine the MIC of colistin sulfate [E. coli NCTC 13846 (colistin resistant) and E. coli ATCC 25922 (colistin susceptible) were used in all MIC experiments as the quality control strains.All the presumptive colistin-resistant  sulfate . To stanmcr genes  was used to screen all the presumptive colistin-resistant E. coli (n = 105) collected from MacConkey agar plates supplemented with colistin sulfate [mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes were E. coli NCTC 13846, E. coli KP37 [E. coli 2013-SQ352 [E. coli DH5α [Salmonella 13-SA01718 [E. coli ATCC 25922 (colistin susceptible) was also included during PCR as a negative control. As an external reference control, GeneRuler 100 bp Plus DNA Ladder  was used in the first and last well of each gel.A previously described method based on multiplex PCR to determine the presence of five plasmid-mediated  sulfate . The posoli KP37 , E. coli13-SQ352 , E. colioli DH5α , and Sal-SA01718 , respect10 CFU of coliforms per gram of feces from before to after treatment were analyzed by a two-tailed unpaired parametric t-test in GraphPad Prism 8.1.1 . The effect of the three different treatments on the growth of pigs was analyzed as the average daily growth with a linear mixed model with individual-level growth from the starting weight to the end-weight as a dependent variable. The explanatory variable in the model was the treatment group (G1–G3), while the pen, nursery unit, and rotation batch were included as a random effect. The weight of each individual pig when entering the nursery unit was included as the covariate in the model. The growth data were recently published in a report by SEGES (in Danish) [Changes in the log Danish) , communiE. coli or co-resistance to other commonly used drugs to treat pigs. Surprisingly, an antidiarrheal feed to pigs around weaning was shown to result in higher weight gain than colistin treatment; however, further studies are needed to explain this observation. The current study has shown that treatment with colistin in the dose and time combinations used did not result in a significant increase in the average colistin MIC values in


To detect the expression levels of microRNA-200a/b (miR-200a/b) in tumor tissues and serum of patients with epithelial ovarian cancer (EOC) and to explore its clinical significance.  A retrospective selection of 30 cases of benign ovarian disease or healthy physical examination (control group) and 55 cases of EOC patients. Real-time quantitative PCR was used to detect the expression level of miR-200a/b in tumor tissues and serum, and the miR-200a/b expresses relevance in the two types of samples were evaluated at the same time. Receiver operating characteristic curve (ROC) and Kaplan-Meier survival analysis were used to evaluate the diagnostic value of miR-200a/b expression and its influence on prognosis, respectively. P < 0.001), and there was a significant positive correlation between serum and tissue miR-200a/b expression . ROC analysis showed that the expression of serum miR-200a/b can distinguish EOC patients from the control group. In addition, there were significant differences in the TNM stage, tumor differentiation, and lymph node metastasis between the miR-200a/b high- and low-expression groups (P < 0.05). Kaplan-Meier survival analysis found that the overall survival and disease-free survival of patients with high miR-200a/b expression were shorter than those of patients with low miR-200a/b expression (P < 0.05).  The serum and tissue miR-200a/b expression levels in EOC patients were higher than those in the control group ( Upregulation of miR-200a/b expression is a common molecular event in EOC patients, and miR-200a/b can be used as a noninvasive biomarker for the diagnosis and prognosis of EOC. Ovarian cancer is one of the three most common female malignancies, with a fatality rate ranking first among female malignancies and a 5-year survival rate of about 48% . EpithelMicroRNA (miRNA) is a noncoding RNA with a length of 18-25 nucleotides, which can complementally bind to the 3′ untranslated region (UTR) of the target messenger RNA (mRNA), participating in the process of regulating gene transcription and translation, thereby regulating gene expression . Over thStudies have found that the miR-200 family is abnormally expressed in EOC and a variety of malignant tumors , 10. AccThis study was approved by the Ethics Committee of Air Force Medical University Tangdu Hospital, and written informed consent was obtained from each participant. All specimens were handled and made anonymous according to the ethical and legal standards.The study subjects included 55 patients with EOC and 30 patients with benign ovarian diseases or healthy physical examinations, all of whom were treated at Tangdu Hospital of Air Force Medical University from January 2016 to December 2020. The age of EOC patients is 32-78 years (median age is 48 years); those with benign ovarian disease or health examination are 28-76 years old (median age is 46 years). The selection criteria for EOC patients were as follows: (1) pathologically confirmed patients with EOC by two different pathologists; (2) received no pre- or postoperative treatment such as chemotherapy or radiotherapy; (3) the patients had no previous history of other cancers; (4) the patients were provided with the detailed follow-up and clinical data. Paraffin blocks of ovarian tissues were obtained from EOC patients who have not undergone chemotherapy or patients with benign ovarian diseases. The control tissue and serum samples are from the benign ovarian diseases or healthy patients. And the EOC tissue and serum samples were taken from the EOC patients.μl of serum, add 5 times the volume of QiAzol lysis Reagent, shake and mix, let stand for 5 min and add 3.5 μl miRNeasy Serum/Plasma Spike-In Control, shake, and mix well. The remaining steps are carried out in accordance with the miRNeasy serum/plasma RNA purification kit  instructions. Tissue miRNA extraction followed the steps: take 50-100 mg of tissue sample and follow the miRcute miRNA extraction and isolation kit instructions for miRNA extraction. The amount of extracted miRNA is stored at -80 degrees. Reverse transcription was performed using The PrimeScript RT-PCR reagent kit . PCR amplification was carried out using SYBR Premix Ex TaqTM II (Takara) with ABI PRISM 7900 System (Applied Biosystems). The upstream primers are 5′-TAA CAC TGT CTG GTA ACG ATG T-3′ (miR-200a) and 5′-TAA TAC TGT CTG GTA ACG ATG T-3′ (miR-200b); the downstream primers are the primers provided in the kit. The upstream primer of the internal reference gene U6 is 5′-GTG CTC CCT GCT TCG GCA GCA CAT ATA C-3′, and downstream primer is 5′-AAA AAT ATG GAA CGC TTC ACG AAT TTG-3′. The expression level of miR-200a/b was analyzed by the relative quantitative method of 2-△△CT. Each qRT-PCR experiment was repeated in triplicate for each sample.The miRNeasy RNA purification kit  was purchased from Qiagen, USA, the miRNA extraction and isolation kit  was purchased from Beijing Tiangen Co., Ltd., the SYBR PrimeScript RT-PCR kit  was purchased from Takara, Japan, and the 7900 QPCR instrument was purchased from ABI, USA. Serum miRNA extraction followed the steps: taking 5 ml of blood sample and centrifuging at 3000 r/min for 10 min. Then, take 200 t-test was used for the pairwise comparison. The χ2 test was used for the count data comparison. Pearson and Spearman's test was used for correlations analysis. The Kaplan-Meier method was used for the survival analysis, and the log-rank test was performed. The receiver operating characteristic curve (ROC) and the area under the ROC curve (AUC) were used to evaluate the diagnostic significance of miRNA. P < 0.05 was considered to indicate a statistically significant difference.Schapiro-Wilk test was used to check the normality distribution of the data before performing statistical analysis. Statistical analysis was performed with the GraphPad Prism 8.0 . The measurement data was expressed by the mean standard deviation. The two-sample Student's P < 0.001, R2 = 0.8212, P < 0.001, P < 0.001, R2 = 0.9005, P < 0.001, The qPCR was performed to detect serum miR-200a/b expression levels in all participants. QPCR results showed that serum miR-200a and miR-200b levels in EOC patients are significantly higher than the control group  . The patient ratio of TNM stages III and IV in the miR-200a/b high-expression group was significantly higher than that in the miR-200a/b low-expression group (P = 0.006 and 0.003). And the ratio of well tumor differentiation in the miR-200a/b high-expression group was also higher than that in the miR-200a/b low-expression group (P = 0.003andP = 0.008). In addition, the incidence of lymph node metastasis in patients with low miR-200a/b expression was significantly lower than that in patients with high miR-200a/b expression (P = 0.021 and 0.013), and the incidence of ascites in patients with low miR-200a/b expression was also significantly lower (P = 0.014 and 0.026).Using the Youden's index of 0.55 and 0.60 for miR-200a and miR-200b, respectively, EOC patients were divided into miR-200a/b high-expression and low-expression groups, and the differences in clinical parameters between the two groups were compared. As shown in Tables P = 0.0047 and P = 0.0187)   and disease-free survival (DFS) of patients with miR-200a high expression were significantly shortened . It is paired with the 3′ untranslated region (3′UTR) of DCAMKL-1 target gene mRNA through the 5′ seed sequence and is responsible for regulating epithelial-mesenchymal transition . PendlebHowever, our study did not find that the level of plasma miRNA-200b in EOC patients is related to the level of CA125. We found that high expression of miR-200b is associated with TNM stage, tumor differentiation, lymph node metastasis, and the incidence of ascites. Through ROC curve analysis, miR-200b expression is helpful to the diagnosis of EOC, with good specificity and sensitivity. Further survival analysis found that the high expression of miR-200a/b was associated with a significant reduction in OS and DFS in EOC patients, indicating that plasma miRNA-200a/b high levels have good EOC diagnostic and prognostic value and can be used as molecular markers for early diagnosis and prognostic prediction of EOC patients. Consistent with our results, it was found that miR-200a, miR-200b, and miR-200c were overexpressed in women with poor OS . MeanwhiIn conclusion, upregulation of miRNA-200a/b expression is a common molecular event in EOC patients, and serum miR-200a/b might serve as a reliable and noninvasive biomarker for the early diagnosis and prognosis prediction of EOC.


Biological replicate and pooling RNA-seq revealed 1870 and 1618 lncRNAs, respectively. Using edgeR, we determined that 1512 genes and 220 lncRNAs, 2240 genes and 127 lncRNAs were differentially expressed in biological replicate and pooling RNA-seq, respectively. After target gene prediction, we found that ACSL3 was cis-targeted by lncRNA TCONS-00052400 and could activate the conversion of long-chain fatty acids. In addition, lncRNA TCONS_00041740 cis-regulated gene ACACB regulated the rate-limiting enzyme in fatty acid oxidation. Since these genes have necessary functions in fat metabolism, the results imply that the lncRNAs detected in our study may affect backfat deposition in swine through regulation of their target genes. Our study explored the regulation of lncRNA and their target genes in porcine backfat deposition and provided new insights for further investigation of the biological functions of lncRNA.Adipose is an important body tissue in pigs, and fatty traits are critical in pig production. The function of long non-coding RNA (lncRNA) in fat deposition and metabolism has been found in previous studies. In this study, we collected the adipose tissue of six Landrace pigs with contrast backfat thickness (n Fat deposition is an important biological process in pig growth. Fatty traits are critical in pig production and are closely related to pork quality as well as the production efficiency and reproductive traits of pigs . PorcineBackfat deposition is a complex biological process influenced by multiple genes and epigenetic factors, including long non-coding RNA (lncRNA). In recent years, there have been an increasing number of studies on lncRNA function related to fat development. The regulation of lncRNA on fat has been reported in various species, such as humans, mice, and cattle. For instance, Alvarez et al.  used de With the rapid development of high-throughput sequencing technology, transcriptome sequencing has been widely used in animal studies. Pooling and biological replicate are two conventional methods used in RNA-seq experiments. Pooling RNA-seq refers to mixing individuals in the same treatment group into one pool for sequencing, which is more efficient, less time-consuming, and incurs lower cost when there are many samples . BiologiIn this study, we performed pooling and biological replicate RNA-seq for three pairs of Landrace pigs with contrast phenotypes of backfat thickness. We identified the differentially expressed genes and lncRNAs between the extremely high- and low-backfat pigs. Combining the results of two methods, we obtained the key genes and lncRNAs that affect backfat deposition in the Landrace pigs. Some novel genes and lncRNAs involved in lipid metabolism are provided for decreasing swine backfat deposition in pig breeding. In addition, we also compared the efficiency of the two RNA-seq methods and the accuracy of their results, which provides new ideas for future experimental design in livestock. p-value = 1.44 × 10−3. The experimental population phenotype and selection criteria were the same as those previously reported [A Landrace female pig resource population was used in this study. We used real-time B-mode ultrasonography  to measure the backfat thickness between the last 3rd and 4th ribs. Combined with pedigree information, we selected three full-sib Landrace pairs with contrast backfat thickness from 132 female Landrace pigs. Three of the pigs with extremely high-backfat thickness formed the high group  and three with extremely low-backfat thickness formed the low group . There was significant difference between the two groups  and the other comprised pigs with extremely low backfat (BL). Then, pooling RNA-seq was performed using these two groups. In biological replicate strand-specific library RNA-seq, there were three independent individuals with similar phenotypes in both BH and BL groups. We identified the differentially expressed genes and lncRNAs in the two-method results and then analyzed their function in fat development. We also explored the specific expressed genes and lncRNAs in different phenotype groups based on the two RNA-seq. The experimental design is shown in We used a total RNA extraction kit  to extract the total RNAs of all the samples, according to the manufacturer’s recommendations, and the quality of the extract was checked by a 2100 Bioanalyzer . After quality control, a library was prepared using the total RNAs. The sequencing was performed by Biomarker Technologies, Inc.  in an Illumina Hiseq 2500 sequencer . The raw data obtained from the sequencing were trimmed using Trimmomatic software . Clean dhttp://asia.ensembl.org/Sus_scrofa/Info/Index, accessed on 20 June 2021) and indexed with Bowtie2 (v 2.3.0) [The pig reference genome (Sscrofa 11.1) and annotation files were downloaded from the Ensembl database (v 2.3.0) . Clean rv 2.3.0)  alignmenv 2.3.0)  was used−5 [−5 were filtered out [High stringency was used to filter out putative lncRNAs from the RNA-seq assembled transcripts. The information in the assembled transcripts from the Cuffmerge results included the class code of each transcript, such as ‘=’, ‘’., ‘c’, ‘j’, ‘e’, ‘i’, ‘o’, ‘p’, ‘r’, ‘u’, ‘x’, and ‘s’. The transcripts with non-coding potential class codes of ‘j’, ‘i’, ‘o’, ‘u’, ‘x’, and ‘=’ were reserved  and all −5 . Then weered out . Similarered out .We used htseq software (v 0.7.2)  to counthttp://www.geneontology.org, accessed on 20 June 2021) [http://www.genome.jp/kegg, accessed on 20 June 2021) [p-values < 0.05 were considered as significantly enriched, and the results were plotted using the ggplot2 [Gene Ontology   is the ine 2021)  is a genne 2021)  and KOBAne 2021) , respect ggplot2  R packagTo investigate the function of the differentially expressed lncRNAs, we searched their nearby genes and considered them as potential targets of lncRNAs. It has been reported that the main function of lncRNA is to regulate protein-coding genes through cis- and trans-regulation ,33. The All data were reported as mean ± standard error of mean. In the edgeR package, we used the qCML  method to estimate the dispersion and the exactTest function for differential expression analysis; the FDR method was used to adjust the p values. An FDR < 0.05 was considered significant.® 480 Real-Time PCR System . Primers used for quantification were designed by Primer-Premier (v6.0) and Primer-BLAST on the NCBI website . RT-PCR reactions were performed in a final volume of 20 μl with the Roche SYBR Green PCR Kit (Roche), according to the manufacturer’s instructions. Pig GAPDH was used as an internal standard to correct the cDNA input. Triplicate RT-qPCRs were performed for each cDNA and the average Ct was used for further analysis. The PCR program was run at 95 °C for 3 min and then 45 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 1 min. The melting curve was then run for 65–95 °C. Relative quantification values were calculated using the 2−ΔΔCt method.Total RNA was extracted from the fat tissues and converted into cDNA using the TaKaRa PrimeScript™RT reagent Kit , following the manufacturer’s protocol. cDNA samples were analyzed with real-time reverse transcriptase (RT)-PCR using the Light CyclerIn the pooling RNA-seq, a total of 15.53 and 14.30 Gb of clean data were separately generated in the extremely high- and low-backfat thickness groups. The guanine-cytosine (GC) contents of the BH and BL group were 51.38% and 49.37%, respectively, and their Q30 were 92.53% and 92.62%, respectively. Furthermore, the results of the biological replicate RNA-seq were 16.20, 16.32, 17.34, 16.24, 16.07, and 16.50 Gb in the six samples. The average of the GC content was between 50.71% and 55.55%, and Q30 ranged from 93.32% to 94.27%. These results showed that the quality of the two libraries of pooling RNA-seq and the six libraries of biological replicate RNA-seq was suitable for subsequent data analysis.Next, the clean reads were aligned to the reference genome (Sus scrofa 11.1) using Tophat v2.1.1. In pooling RNA-seq, more than 77.82% of the clean reads were uniq mapped. In all samples of biological replicate RNA-seq, the uniq mapped ratios were larger than 83.77%. The summary of the sequencing data is shown in After using cufflinks and cuffmerge, we obtain a combined GTF file containing 146,397 transcripts. Then we followed stringent criteria and created a pipeline to identify lncRNAs, as shown in In the pooling RNA-seq, 2240 genes and 127 lncRNAs  were difIn addition, to further explore the difference in gene expression between the extremely high- and low-backfat thickness groups, we analyzed the specific expressed genes and lncRNAs in the two groups. There were 643 SEGs and 136 SELs in the BH group and 892 DEGs and 177 DELs in the BL group in pooling RNA-seq b,c.In the biological replicate RNA-seq, we performed the same analysis as that used for the pooling RNA-seq. We found that 1512 genes and 220 lncRNAs  were difMoreover, we identified 620 genes and 133 lncRNAs specific expressed in the BH group and 607 genes and 118 lncRNAs specific expressed in the BL group based on the result of biological replicate RNA-seq b,c.We used the differentially expressed genes detected in the pooling and biological replicate RNA-seq methods to perform functional enrichment analysis, respectively. The Gene Ontology results of DEGs based on the pooling RNA-seq are shown in In the biological replicate RNA-seq, a total of 977 DEGs were annotated in GO terms. We found many terms directly related to fat development and metabolism, especially in the BP class, including ‘tricarboxylic acid cycle’, ‘triglyceride homeostasis’, and ‘triglyceride catabolic process’. Furthermore, there were four mRNAs enriched to ‘positive regulation of triglyceride catabolic process’. This GO term was also significant but not within the top ten; therefore, it is not displayed in In addition, we used the specific expressed genes from the pooling and biological replicate RNA-seq to perform the functional enrichment analysis, respectively. These results are shown in We compared the library construction quality of the two sequencing methods . We founIn the present study, we compared the expression of the putative lncRNAs with the genes. We found that the lncRNAs tended to be expressed at a lower level than the protein-coding genes in both the pooling and biological replicate RNA-seq a,b. A toIn the functional enrichment analysis of DEGs , both biTo further understand the potential function of lncRNA in fat deposition, we predicted the target genes of the lncRNAs. To increase the reliability of our results, we selected the DELs that were shared by the two sequencing methods and then predicted their target genes. We searched the nearby coding genes around 100 kb up- and downstream from the 39 DELs and found 76 pairs of cis-regulatory relationships between 39 DELs and 67 genes . p < 0.05). They were also considered as potential target genes of these DELs. We used 39 DELs and their target genes to draw a picture of the regulatory network to investigate the relationship between them  and 5 DELs  to perform qPCR. As shown in We randomly selected 5 DEGs  is located 64 kb downstream of TCONS-00052400. At the beginning of fatty acid metabolism, long-chain acyl-CoA synthetase (ACSL) can activate the conversion of long-chain fatty acids to fatty acyl-CoA [ACSL family contains five different isoforms, including ACSL3, which play different roles in lipid metabolism. Some previous studies showed that knockdown of ACSL3 may significantly reduce the activity of several lipid-producing transcription factors such as peroxisome proliferator-activated receptor-γ and sugar response element binding protein, and then regulate the fat production process in the liver [ACSL3 can also affect the secretion of very low-density lipoproteins (VLDL) by promoting the synthesis of lecithin [ACACB (acetyl-CoA carboxylase beta) is a differentially expressed gene that is cis-regulated by the lncRNA TCONS-00041740, which is located on the antisense strand of ACACB. Moreover, in the KEGG enrichment analysis, ACACB was involved in the ‘pyruvate metabolism’ pathway. Based on previous studies, we knew that ACACB is the rate-limiting enzyme in fatty acid oxidation [ACACB knockout mice, continuous fatty acid oxidation increases insulin sensitivity, and feeding them a high fat/high carbohydrate diet is more likely to cause obesity and diabetes [ACACB was shown to be a marker gene for childhood obesity.As a kind of non-coding RNA, the main role of lncRNA is to regulate their target genes: cis-regulating nearby protein-coding genes and trans-regulating distant protein-coding genes. We determined 67 cis-target genes and 116 trans-target genes regulated by DELs. The two target genes we focused on were acyl-CoA . The ACShe liver . Furtherlecithin . ACACB  was specific expressed in the BL group. Phospholipase C is a class of glycerol phospholipid hydrolases, hydrolyzing the glycerol phosphate C3 site [PLCB2 is a phosphodiesterase that catalyzes the hydrolysis of phosphatidylinositol 4, 5-bisphosphate to the secondary messengers inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol. In addition, the gene PLA2G12B was specific expressed in the BH group. PLA2G12B (phospholipase A2 group XIIB) is encoded by this gene and belongs to the phospholipase A2 (PLA2) group of enzymes, which plays a role in lipid hydrolysis by releasing free fatty acids and lysophospholipids [PLA2G12B decreases the amount of serum triglyceride (TG)-rich VLDL particles secreted by the liver, resulting in a reduction in TG content [PLA2G12B can also participate in the pathogenesis of idiopathic membranous nephropathy (iMN) by regulating lipid metabolism [PLA2G12B-null mice had obvious accumulation of large lipid droplets in the liver, displaying the fatty liver phenotype. These results indicate that the genes specific expressed in the high or low backfat groups may also have a certain regulatory effect on lipid metabolism.Some genes were specific expressed in one of the two groups. For example,  C3 site . The proholipids . Studies content . PLA2G12tabolism . In a prtabolism , PLA2G12During pig growth, the excessive development of adipose tissue leads to an excessive accumulation of lipids, which affects the carcass quality of pigs. Overweight pigs show low lean meat rates, low feed conversion rates, and slow growth , while oACACB, and TCONS-00052400 to ACSL3, may have an effect on fat deposition. These results can provide useful information for understanding the regulation of fat deposition by lncRNA in pigs. In this study, we identified 1512 DEGs and 220 DELs between pigs with extremely high- and low- fat deposition traits in biological replicate RNA-seq, and 2240 DEGs and 127 DELs in pooling RNA-seq, respectively; the former revealed more genes, but the two methods were similar in terms of gene functional enrichment. After the analysis of potential cis- and trans- target genes, we found 183 genes that could be regulated by DELs. Through further functional analysis, we found that two pairs of potential targeting relationships between lncRNAs and genes, TCONS-00041740 to